Sensor device for interference and plasmon-waveguide/interference spectroscopy

ABSTRACT

Methods for measuring a property of a sample material present at an interface of an emerging medium in a spectroscopic device include a sensor ( 10 ) featuring at least one dielectric member ( 14 ) disposed upon a entrant medium ( 12 ), with the dielectric member being of an optical thickness selected to produce an observable interference effect for an incident angle of light below the critical angle of total internal reflection at the interface with the dielectric member ( 14 ). The dielectric member ( 14 ) of the sensor device ( 10 ) may be modified and functionalized for binding of a sample analyte by disposing a sensing surface area ( 38, 40, 42 ) upon it. Moreover, an metal layer ( 18 ) may be added to an entrant medium ( 20 ) to produce a sensor ( 16 ) that generates observable optical phenomena both above (resonance) and below (interference) a given critical angle.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims benefit of priority under 35 U.S.C. 119(e) toInternational Application No. PCT/US03/30694 filed on Sep. 30, 2003, theentire contents of which are incorporated herein by reference.

U.S. GOVERNMENT RIGHTS

This invention was made with Federal Government support under ContractNumber MCB-9904753 awarded by the National Science Foundation andContract Number GM-59630 awarded by the National Institutes of Health.The Government has certain rights in the invention.

BACKGROUND OF THE INVENTION

1. Field of the Invention

This invention relates in general to optical sensor chips and moreparticularly to an optical sensor chip that utilizes the phenomenon ofoptical interference to spectroscopically characterize the properties ofmolecules below the critical angle of incidence.

2. Description of the Related Art

Surface plasmon resonance is a phenomenon used in many analyticalapplications in metallurgy, microscopy, and chemical and biochemicalsensing. Along with optical techniques such as ellipsometry, multipleinternal reflection spectroscopy, and differential reflectivity, SPR isone of the most sensitive techniques to surface and interface effects.This inherent property makes SPR well suited for nondestructive studiesof surfaces, interfaces, and very thin layers. The SPR phenomenon hasbeen known for decades and the theory is fairly well developed. Simplystated, a surface plasmon is an oscillation of free electrons thatpropagates along the surface of a conductor. The phenomenon of surfaceplasmon resonance occurs under total internal reflection conditions atthe boundary between substances of different refractive indices, such asglass and water solutions. When an incident light beam is reflectedinternally within the first medium, its electromagnetic field producesan evanescent wave that crosses a short distance (in the order ofnanometers) beyond the interface with the second medium. If a thin metalfilm is inserted at the interface between the two media, surface plasmonresonance occurs when the free electron clouds in the metal layer (theplasmons) absorb energy from the evanescent wave and cause a measurabledrop in the intensity of the reflected light at a particular angle ofincidence that depends on the refractive index of the second medium.

Typically, the conductor used for SPR spectrometry is a thin film ofmetal such as silver or gold; however, surface plasmons have also beenexcited on semiconductors. The conventional method of exciting surfaceplasmons is to couple the transverse-magnetic (TM) polarized energycontained in an evanescent field to the plasmon mode on a metal film.The amount of coupling, and thus the intensity of the plasmon, isdetermined by the incident angle of the light beam and is directlyaffected by the refractive indices of the materials on both sides of themetal film. By including the sample material to be measured as a layeron one side of the metallic film, changes in the refractive index of thesample material can be monitored by measuring changes in the surfaceplasmon coupling efficiency in the evanescent field. When changes occurin the refractive index of the sample material, the propagation of theevanescent wave and the angle of incidence producing resonance areaffected. Therefore, by monitoring the angle of incidence at a givenwavelength and identifying changes in the angle that causes resonance,corresponding changes in the refractive index and related properties ofthe material can be readily detected.

As those skilled in the field readily understand, total reflection canonly occur above a particular critical incidence angle if the refractiveindex of the incident or entrant medium (typically a prism or grating)is greater than that of the emerging medium. In practice, totalreflection is observed only for incidence angles within a range narrowerthan from the critical angle to 90 degrees because of the physicallimitations inherent with the testing apparatus. Similarly, for systemsoperating with variable wavelengths and a given incidence angle, totalreflection is also observed only for a corresponding range ofwavelengths. This range of incidence angles (or wavelengths) is referredto as the “observable range” for the purpose of this disclosure.Moreover, a metal film with a very small refractive index (as small aspossible) and a very large extinction coefficient (as large as possible)is required to support plasmon resonance. Accordingly, gold and silverare appropriate materials for the thin metal films used in visible-lightSPR; in addition, they are very desirable because of their mechanicaland chemical resistance.

Thus, once materials are selected for the prism, metal film and emergingmedium that satisfy the described conditions for total reflection andplasmon resonance, the reflection of a monochromatic incident beambecomes a function of its angle of incidence and of the metal'srefractive index, extinction coefficient, and thickness. The thicknessof the film is therefore selected such that it produces observableplasmon resonance when the monochromatic light is incident at an anglewithin the observable range.

The classical embodiments of SPR devices are the Kretschmann and Ottoprism or grating arrangements, which consist of a prism with a highrefractive index n (in the 1.4-1.7 range) coated on one face with a thinfilm of metal. The Otto device also includes a very thin air gap betweenthe face of the prism and the metal film. In fact, the gap between theprism (or grating) and the metal layer, which is in the order ofnanometers, could be of a material other than air, even metal, so longas compatible with the production of observable plasmon resonance in themetal film when the monochromatic light is incident at an angle withinthe observable range.

Similar prior SPR devices are based on the phenomenon of long-rangesurface plasmon resonance, which is also generated with p-polarizedlight using a dielectric medium sandwiched between the incident mediumand a thinner metal layer (than in conventional SPR applications). Themetal film must be sufficiently thin and the dielectric and emergentmedia must be beyond the critical angle (i.e., having refractive indicessmaller than the refractive index of the entrant medium) so that theysupport evanescent waves to permit the simultaneous coupling of surfaceplasmons at the top and bottom interfaces of the thin metal layer (i.e.,to permit excitation of surface waves on both sides of the thin metalfilm). This condition is necessary in order for the phenomenon oflong-range surface plasmon resonance to occur. For a given set ofparameters, the distinguishing structural characteristic betweenconventional surface plasmon resonance and long-range surface plasmonresonance is the thickness of the metal film and of the inner dielectricfilm (the latter not being necessary for conventional SPR). In theconventional technique, the metal film must be sufficiently thick andmust be placed either directly on the entrant medium (i.e., prism orgrating) or on a dielectric film which is too thin to allow excitationof the surface bound waves on both metal surfaces, to produce observableplasmon resonance when a monochromatic light is incident at an anglewithin the observable range.

In long-range surface plasmon resonance (LRSPR), in contrast, the metalfilm must be placed between two dielectric media that are beyond thecritical angle so that they support evanescent waves, and must be thinenough to permit excitation of surface waves on both sides of the metalfilm. The specific thickness depends on the optical parameters of thevarious components of the sensor in question, but film thicknesses inthe order of 45-55 nm for gold and silver are recognized as critical forconventional SPR, while no more than about half as much (15-28 nm) canbe used for LRSPR. It is noted that the thickness required to supporteither form of surface plasmon resonance for a specific system can becalculated by one skilled in the art on the basis of the system'soptical parameters.

As well understood by those skilled in the art, the main criterion for amaterial to support SP waves is that it have a negative real dielectriccomponent, which results from the refractive and extinction propertiesmentioned above for the metal layer. The surface of the metal film formsthe transduction mechanism for the SPR device and is brought intocontact with the sample material to be sensed at the interface betweenthe metal film and the emerging medium contained in a cell assembly.Monochromatic light is emitted by a laser or equivalent light sourceinto the prism or grating and reflected off the metal film to an opticalphotodetector to create the sensor output. The light launched into theprism and coupled into the SP mode on the film is p-polarized withrespect to the metal surface where the reflection takes place. In allthese prior-art devices and techniques, only p-polarized light iscoupled into the plasmon mode because this particular polarization hasthe electric field vector oscillating normal to the plane that containsthe metal film. This is sometimes referred to as transverse-magnetic(TM) polarization.

As mentioned, the surface plasmon is affected by changes in thedielectric value of the material in contact with the metal film. As thisvalue changes, the conditions necessary to couple light into the plasmonmode also change. Thus, SPR is used as a highly sensitive technique forinvestigating changes that occur at the surface of the metal film. Inparticular, over the last several years there has been a keen interestin the application of surface plasmon resonance spectroscopy to studythe optical properties of molecules immobilized at the interface betweensolid and liquid phases.

The ability of the SPR phenomenon to provide information about thephysical properties of dielectric thin films deposited on a metal layer,including lipid and protein molecules forming proteolipid membranes, isbased upon two principal characteristics of the SPR effect. The first isthe fact that the evanescent electromagnetic field generated by the freeelectron oscillations decays exponentially with penetration distanceinto the emergent dielectric medium; i.e., the depth of penetration intothe material in contact with a metal layer extends only to a fraction ofthe light wavelength used to generate the plasmons. This makes thephenomenon sensitive to the optical properties of the metal/dielectricinterface without any interference from the properties of the bulkvolume of the dielectric material or any medium that is in contact withit. The second characteristic is the fact that the angular (orwavelength) position and shape of the resonance curve is very sensitiveto the optical properties of both the metal film and the emergentdielectric medium adjacent to the metal surface. As a consequence ofthese characteristics, SPR is ideally suited for studying bothstructural and mass changes of thin dielectric films, including lipidmembranes, lipid-membrane/protein interactions, and interactions betweenintegral membrane proteins and peripheral, water-soluble proteins. SeeSalamon, Z., H. A. Macleod and G. Tollin, “Surface Plasmon ResonanceSpectroscopy as a Tool for Investigating the Biochemical and BiophysicalProperties of Membrane Protein Systems. I: Theoretical Principles,”Biochim. et Biophys. Acta, 1331: 117-129 (1997); and Salamon, Z., H. A.Macleod and G. Tollin, “Surface Plasmon Resonance Spectroscopy as a Toolfor Investigating the Biochemical and Biophysical Properties of MembraneProtein Systems. II: Applications to Biological Systems,” Biochim. etBiophys. Acta, 1331: 131-152 (1997).

In U.S. Pat. No. 5,991,488, herein incorporated by reference, wedisclosed new thin-film interface designs that couple surface plasmonand waveguide excitation modes. The technique, defined as coupledplasmon-waveguide resonance (CPWR), is based on the concept of couplingplasmon resonances in a thin metal film with the waveguide modes in adielectric overcoating. Accordingly, a metallic layer, typically eithergold or silver, is used with a prism so as to provide a surface plasmonwave by conventional SPR (or waves by long-range SPR) and is furthercovered with a solid dielectric layer characterized by predeterminedoptical parameters. The dielectric member inserted between the metalfilm and the emergent medium is selected such that coupledplasmon-waveguide resonance effects are produced within an observablerange.

The emergent dielectric medium is then placed in contact with this soliddielectric layer. As disclosed in the patent, we found that theadditional layer of dielectric material functions as an opticalamplifier that produces an increased sensitivity and enhancedspectroscopic capabilities in SPR. In particular, the added dielectriclayer makes it possible to produce resonance with either s- orp-polarized light. In addition, the added dielectric protects the metallayer and could be used as a matrix for adsorbing and immobilizing thesensing materials in CPWR-based biosensor applications, as disclosed inPCT Application Serial No. US03/0273, which is herein incorporated byreference.

Nonetheless, a remaining drawback of SPR and CPWR is that one is limitedto measuring resonance effects above the critical angle for totalinternal reflectance. In other words, conventional SPR and CPWR arelimited to the probing of optical properties observable in thereflectance mode of a spectrometer. Moreover, conventional CPWR sensorscontain a dielectric layer or member that is on the order of nanometersin thickness, which can be too thin and fragile for certain surfacemodifications (e.g., etching techniques) and thus limit the versatilityof the sensor.

Therefore, a need still exists for a new and useful sensor chip thatovercomes some of the problems and shortcomings of the art.

SUMMARY OF THE INVENTION

The invention relates in general to a new sensor device that utilizesoptical interference for measuring properties of molecules and/or thinfilms at surfaces and interfaces. More specifically, the sensor deviceof invention is able to probe the optical properties of surfaces andinterfaces both in the reflectance and transmittance modes by utilizinga dielectric layer or member of appropriate optical thickness togenerate interference effects that are observable below the criticalangle of total internal refection. This is in contrast with conventionalSPR and CPWR spectroscopy, which are essentially limited to measurementsof resonance effects above the critical angle. Moreover, a metal orsemiconductor layer can be added to the sensor of the invention suchthat both interference effects and coupled-plasmon waveguide resonanceeffects can be measured for a given analyte.

In a preferred embodiment, the sensor device includes an entrant mediumin optical contact with a dielectric member (e.g., SiO₂) that is about100 microns in thickness. A sample solution is placed on the dielectricmember and an incident angle of light below the critical angle for totalinternal reflection is reflected by and transmitted through thedielectric member, thereby producing observable interference effects inboth reflectance and transmittance modes.

In another preferred embodiment, the device includes an entrant mediumand a metal (or semiconductor) layer or layers with appropriate opticalparameters and thickness (e.g., gold or silver with a thickness between25-85 nm) in which surface plasmons can be generated by light. Adielectric member is in optical contact with the metal layer andfunctions both as a plasmon waveguide and as an optical interferencesystem. Thus, especially sensitive spectroscopy can be performed becauseeither optical interference effects or plasmon-waveguide resonanceeffects (or both) can be measured at incident angles of light both belowand above the critical angle.

One of the main goals of this invention is the development of sensordevices that expand the observable range of optical phenomena that canbe used to characterize molecules during spectroscopy. To that end, theinvention further relates to a sensor unit for use in a system based onCPWR technology that includes a dielectric substrate having one or moresensing areas that contain at least one functional group supporting bi-or polyfunctional molecules capable of specific binding with moleculespresent in a sample analyte. Preferably, coupling between the bi- orpolyfunctional molecules and the molecule(s) in the sample isselectively reversible such that the bi- or polyfunctionalmolecule-functionalized sensing areas can be regenerated, permittingrepeated use of the functionalized sensor unit.

The sensor of the present invention differs from previous SPR and CPWRsensors in several key respects. First, the invention features adielectric member that is approximately an order of magnitude thickerthan that found on previous sensors (i.e., 10-1000 microns thick asopposed to 50-1200 nanometers). The inventors discovered that adielectric material of an appropriate optical thickness (defined in theart as the product of refractive index and geometric thickness of thedielectric medium) can be used to generate optical interference effects,which can be measured in transmittance or reflectance modes at incidentangles of light below the critical angle during spectroscopy. Second,CPWR sensors are limited to either p- or s-polarized light becauseplasmon oscillations occur only in the p and s directions; sensors ofthe present invention are not so limited. Third, the thick dielectricmember allows many forms of surface modification to be practiced withless risk of damage to the sensor. Fourth, when a metal or semiconductorlayer is added to the interference-causing dielectric layer of thepresent invention, the resulting sensor produces observable spectra atany angle of incidence (i.e., interference effects below the criticalangle and coupled-plasmon waveguide resonance effects above the criticalangle for total internal reflection).

Thus, an objective of the invention is to provide a sensor forinvestigating the properties of molecules at surfaces and interfacesthat is capable of generating observable interference effects.

Another object of the invention is to provide a sensor device thatgenerates observable spectral effects below the critical angle of totalinternal reflection for a given interface.

Another goal of the invention is to provide a method for measuring aproperty of a sample material present at an interface of an emergingmedium that utilizes transmitted light in addition to reflected light.

Another important objective is to provide a sensor that is suitable fortesting samples using monochromatic light with an angle of incidenceranging from 0 to 90 degrees.

Yet another goal of the invention is to provide a technique that makesit possible to achieve high throughput testing applications with anefficient, practical and economically feasible implementation.

Still another objective is to provide sensor devices that are suitablefor direct incorporation with existing CPWR spectroscopic and otherspectroscopic instruments.

Therefore, according to these and other objectives, the presentinvention pertains to novel and improved sensors featuring dielectricmembers of appropriate optical thickness such that observable opticalinterference effects can be measured during spectroscopy.

Various other purposes and advantages of the invention will become clearfrom its description in the specification that follows and from thenovel features particularly pointed out in the appended claims.Therefore, to the accomplishment of the objectives described above, thisinvention consists of the features hereinafter illustrated in thedrawings, fully described in the detailed description of the preferredembodiment and particularly pointed out in the claims. However, suchdrawings and description disclose but one of the various ways in whichthe invention may be practiced.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a schematic view of a coupled plasmon-waveguide resonancespectroscopic tool according to the prior art in an attenuated totalreflection measuring system, wherein a glass prism coated with a 50nm-thick silver layer is protected by a 460 nm-thick Si0 ₂ film.

FIG. 2 is a schematic view of a preferred embodiment of the sensor ofthe invention.

FIG. 3 is a schematic view of a second preferred embodiment of theinvention that includes a metal layer for generating coupled-plasmoneffects.

FIG. 4A illustrates spectra obtained with the sensor device of FIG. 3 inboth transmittance and reflectance modes using p-polarized light.

FIG. 4B illustrates spectra obtained with the sensor device of FIG. 3 inboth transmittance and reflectance modes using and s-polarized light.

FIG. 5A illustrates the reflectance spectra measured above the criticalangle for total internal reflection (see FIGS. 4A, B) using p-polarizedlight. Curve 1 shows the spectrum obtained with a bare sensor in contactwith an aqueous solution (as shown in FIG. 3), whereas curve 2represents the spectrum of the sensor with an additional coating thathas the same optical properties as a typical lipid bilayer membrane.

FIG. 5B illustrates the reflectance spectra measured above the criticalangle for 20 total internal reflection (see FIGS. 4A, B) usings-polarized light. Curve 1 shows the spectrum obtained with a baresensor in contact with an aqueous solution (as shown in FIG. 3), whereascurve 2 represents the spectrum of the sensor with an additional coatingthat has the same optical properties as a typical lipid bilayermembrane.

FIG. 6A illustrates reflectance spectra measured below the criticalangle obtained with the sensor device of FIG. 3 using p-polarized light.Curves 1 and 2 are as described in the legend for FIGS. 5A, B.

FIG. 6B illustrates reflectance spectra measured below the criticalangle obtained with the sensor device of FIG. 3 using s-polarized light.Curves 1 and 2 are as described in the legend for FIGS. 5A, B.

FIG. 7A illustrates transmittance spectra obtained using p-polarizedlight as described in the legend for FIG. 6A.

FIG. 7B illustrates transmittance spectra obtained using s-polarizedlight as described in the legend for FIG. 6B.

FIG. 8 is a schematic view of a third embodiment of the inventionfeaturing dielectric surface functionalization.

DESCRIPTION OF THE PREFERRED EMBODIMENTS

This invention is the result of further development of the CPWR devicesdescribed in our previous U.S. Pat. Nos. 6,421,128; 6,330,387;5,991,488. In general the invention relates to a sensor for measuringthe properties of molecules at surfaces and interfaces that has adielectric layer or member that is of an optical thickness selected toproduce an observable interference effect for an incident angle of lightbelow a critical angle of total internal reflection. Moreover, theinvention involves a sensor that combines the phenomena of opticalinterference and coupling of plasmons into waveguide modes in onesensing device. Thus, the sensor of the invention may be used to measureobservable effects that occur both above and below the critical anglefor total internal reflection.

It should be understood that the dielectric layer of the invention is inaddition to and separate from the sample material or analyte with whichthe invention is used. The sample material at the interface with theemerging medium is often itself dielectric in nature, but its propertiescannot be used to obtain the advantages of the invention without theaddition of a appropriate dielectric layer as disclosed in thisapplication. Therefore, all references to dielectric material pertainonly to the additional layer contemplated herein.

Turning to FIG. 1, a CPWR sensor device 2 of the prior art isillustrated schematically. An entrant medium 4 (in this case, a prism)is coated with a metal layer 6 such as silver or gold about 50 nm inthickness. The metal layer 6 produces plasmons in response to lightwaves generated by the light source at an incident angle ∝. A 460 nmcoating of dielectric material 8 (such as glass) acts as a plasmonwaveguide such that resonance effects can be measured when s- andp-polarized light is used. However, resonance effects are limited toconditions at which the incident angle of light is above the criticalangle for total internal refection.

FIG. 2 shows an embodiment of the invention in which a sensor 10includes an entrant medium 12 in optical contact with a dielectricmember 14 that is about 100 micrometers in thickness. Becauseinterference effects are visible at incident angles below the criticalangle for total internal reflection, changes in surface properties canbe measured. Thus, optical interference effects both in transmittance orreflectance are used to characterize a property of a sample placed uponthe dielectric member 14.

Suitable dielectric materials must be selected to provide an opticalthickness that will produce an observable interference effect for anincident angle of light below the critical angle of total internalreflection. For example, a glass prism coated with a 100micrometer-thick SiO₂ layer (n_(d)=1.4571, k_(d)=0.0030) is suitable topractice the invention with an aqueous analyte (n_(e)=1.33). Otherpossible dielectric materials included, but are not limited to, TiO₂,MgF₂, AL₂O₃, LaF₃, Na₃ALF₆, ZnS, ZiO₂, Y₂O₃, HfO₃, Ta₂O₅, ITO, nitritesor oxy-nitrites of silicone and aluminum, and combinations of these(e.g., layers of TiO₂ and SiO₂).

Turning to FIG. 3, a preferred embodiment of the invention featuring asensor device 16 that includes a metal (or semiconductor) layer 18 orlayers with suitable optical parameters and thickness (e.g., gold orsilver between 25-85 nm in thickness) and in which surface plasmons canbe generated by light is shown. The metal layer 18 is deposited eitherdirectly on an entrant medium 20 (i.e., either a prism or a diffractiongrating) or on a very thin layer (e.g., on the order of a few nanometersthick) of some additional material (not shown) that is used to improveadhesion of the metal layer to the entrant medium surface.

The metal layer 18 is either coated with an emergent medium (dielectricmember 22) or is in optical contact with it. The dielectric member 22may include either one or many layers of different materials and isdesigned in this embodiment to function both as an optical waveguide andan optical interference system. Accordingly, the thickness of thedielectric material is on the order of micrometers in thickness,typically between 10-1000 microns. Thus, one of the main advantages ofthis embodiment of the invention is its high sensitivity and spectralresolution in characterizing the optical properties of small amounts ofmaterials immobilized on surfaces and interfaces for all incident anglesof light between 0 and 90 degrees. Another advantage is that the sensordevice of the invention uses thick and easily replaceable dielectricmembers made from a wide range of materials. Thus, high throughputsensor applications can be readily fabricated.

FIGS. 4A, B through 6A, B illustrate the plasmon resonance andinterference spectra obtained from the device of FIG. 3, either with p-or s-polarized light as indicated in the brief description of thefigures above. As can be seen from the data in these figures, and incontrast with previous sensor designs, the sensor device of theinvention is able to generate p- and s-polarized observable effects bothabove (i.e., resonance effects) and below (i.e., interference effects)the critical angle and can measure interference effects using bothreflectance and transmittance modes.

The device is highly sensitive to changes in the optical properties ofthe interface between the dielectric layer and the emergent medium. Thiscan be demonstrated by comparing spectra obtained with and without anadditional thin film coating. FIGS. 5A and 5B represent spectra measuredabove the critical angle with p- and s-polarized light, respectively,whereas FIGS. 6A and 6B represent reflectance and transmittance spectrameasured below the critical angle. Curves 1 show the spectra obtainedwith a bare sensor in contact with an aqueous solution and curves 2 thesensor with an additional coating that has the same optical propertiesas a typical lipid bilayer membrane. FIGS. 7A (using p-polarized light)and 7B (using s-polarized light) show the transmittance spectra obtainedas described for FIGS. 6A and 6B, respectively.

Choosing materials with the appropriate optical parameters and physicalproperties allows the device to be used in a large variety ofapplications. For example, the sensor of the invention can be used in awide spectral range (from the ultraviolet to the infraredelectromagnetic regions). Furthermore, the micrometer thickness of thedielectric layer allows different forms of surface modification of thesenor chip to be used, including various types of lithographic andetching techniques to create specialty sensors. For example, mini-porearrays within which lipid bilayers can be deposited for high throughputsensors can be created.

Furthermore, the thick dielectric film can also be designed to serveboth as an optical and an electrical device that is capable ofmonitoring simultaneously electrical characteristics and opticalparameters of thin films and interfaces. Also, the device of theinvention can be used to characterize light-absorbing chromophoreswithin different spectral ranges and at multiple wavelengths. Thus, thesensor of the invention can be used in all forms of surface plasmonresonance-based spectrometers and biosensors, as well as in all otheroptical and measurement techniques using surface phenomena.

A biosensor is generally defined as a unique combination of a receptorfor molecular recognition, for example a selective layer withimmobilized antibodies, and a transducer for transmitting the valuesmeasured. Accordingly, a biosensor will detect the change which iscaused in the optical properties of a surface layer due to theinteraction of the receptor with the surrounding medium (such as couldbe detected by SPR, CPWR, or PWI). Thus far, however, work on thedevelopment of biosensors has focused almost exclusively on variousmethods for binding a particular biomolecule to the metal surface of anSPR device. For example, in U.S. Pat. No. 5,492,840, Malmqvist et al.discloses a sensor unit for use in SPR-based systems that features ametal film having several sensing surfaces that have been disposedthereon and functionalized with antibodies and various other chemicalelements.

Biomembranes, such as lipid bilayers, have been described and used tomodify the dielectric member of a SPR and CPWR devices. While this typeof surface modification would also allow the use of the presentinvention in biosensor applications, a great number of chemical orpharmaceutical samples will not be immobilized. Hence, functionalizationof the dielectric layer found on the sensor of the invention providesthe ability to perform interference-based or coupled-plasmonwaveguide/interference spectroscopy (PWI) with wide variety ofbiological and non-biological molecules.

The functionalized sensors of the invention may be made in one piece,for example, from a glass slide (a dielectric material). Accordingly, inone embodiment of the invention, a layer of an organic polymer or ahydrogel forming a so-called basal surface that contains functionalgroups for binding the desired ligands is applied to the surface of theglass slide. These organic polymer or hydrogel layers thus becomesensing surfaces.

For measurements to be carried out, the sensing surfaces have to befunctionalized with different ligands capable of interaction with atarget chemical or molecule. Several routes are available in achievingsuch functionalization. For example, the basal surface may be providedwith a particular ligand during the manufacturing process or a user mayprovide his or her own. The ligands employed are bi- or polyfunctional,meaning that the ligands contain a function that is utilized forimmobilization on a corresponding sensing surface of the dielectriclayer, plus one or more bioselective functions for interaction withmolecules in a sample.

So-called chimeric molecules (bi- or polyfunctional molecules) can beemployed for functionalizing the sensing surfaces of the dielectriclayer. The chimeric molecules comprise one portion binding to the basalsurface, e.g., a dextran-coated sensing surface, and one portion havingan affinity for the biomolecule to be detected. Bi- or polyfunctionalmolecules for use according to the invention may be produced in avariety of ways, e.g., by means of chemical coupling of the appropriatemolecules or molecule fragments, by means of hybridoma techniques forproducing bifunctional antibodies, or by means of recombinant DNAtechniques. This last-mentioned technique involves the fusing of genesequences coding for the structures which are wanted in the product,this product then being expressed in a suitable expression system suchas, e.g., a culture of bacteria. The covalent immobilization of nucleicacids to the sensing surface can then be accomplished as described inSwanson, M. J. et al. “Reactive Polymer-Coated Surfaces for CovalentImmobilization of Nucleic Acids” (describing activated glass surfacesavailable from Amersham Biosciences as a “CodeLink Activated Slide” andformerly available from Surmodics, Inc. as a “3D-Link MicroarraySlide.”).

Chemical coupling of biomolecules or fragments thereof can be performedin accordance with one of the coupling methods that have been developedfor the immobilization of biomolecules (see, for example, Moser, I. etal., Sensors and Actuators B. 7, (1992) 356-362). A suitable reagent is,e.g., SPDP N-succinimidyl 3-(2-pyridylthio)propionate, aheterobifunctional reagent (from Pharmacia AB, Sweden), and with acoupling technique as described by Carlsson et al. Biochem. J. 173:223(1978). In the case of dextran, the chimeric molecule may consist of anantibody against dextran conjugated with a biospecific ligand, e.g., animmunoglobulin.

According to an alternative procedure, a sensing surface is modifiedwith a so-called hapten for binding chimeric molecules to the surface.Thus, for example, a reactive ester surface as described above may bederivatized with a theophylline analog which is then utilized forbinding chimeric molecules. In this case, the chimeric molecule consistsof an antibody against theophylline conjugated with a biospecificligand. This alternative embodiment very clearly reveals the greatversatility attainable with the use of surfaces according to the presentinvention, inasmuch as it is very easy for the user to provide the samesingle basal surface with the desired ligand (e.g., receptor).

Proteins as a base for chimeric molecules (i.e., the surface bindingstructure thereof) must have strong bonds to their low-molecularchemically stable partners on the measuring basic surface. Also, no partthereof should, of course, be capable of interacting with the biosystemsto be analyzed. Thus, in order to control non-specific binding, abiospecific pair derived from a plant, for example, could be used in themeasurement of analytes containing human proteins.

Preferably, the analytical system is such that the binding of thechimeric molecule to the analyte may be reversed under conditionsdiffering from those permitting the binding between the measuringsurface and the chimeric molecule to be broken. In such manner,depending on the conditions, the sensing surface may be regenerated attwo different levels, i.e., either for binding a new analyte, or forre-functionalizing the surface with the same or other chimericmolecules.

It is noted that the effects on the dielectric layer of the inventionare not diminished by the addition of a very thin (1-5 nm) layer of gold(or other metal or a semiconducting material) at the interface with theemerging medium for the purpose of fixating the analyte to the sensingmolecules. Such a combination of properties in one interface permits theconstruction of a durable sensor device with very high sensitivity andan expanded dynamic range of measurements. In other words, the additionof a very thin overcoating of the dielectric layer with a metal orsemiconductor determines the type of chemistry required for surfacemodification and functionalization.

As is known to those skilled in the art, sulfur-bearing compounds caneasily modify metal surfaces (including noble metal surfaces).Long-chain thiols, such as HS(CH₂)_(n)X with n>10, adsorb from solutiononto noble metals and form densely packed oriented monolayers (See, ingeneral, S. Heyse et al. Biochimica et Biophysica Acta 85507 (1998)319-338). The terminal group, X, of the thiol can be chosen from a widevariety of functional groups to interact properly with sample molecules.The choice of functional group determines the properties of suchmodified surfaces and its interaction with sample molecules. Monolayersof alkylthiols (X═CH₃) may serve as hydrophobic supports, allowingsamples to be immobilized by hydrophobic interactions. Alternatively,hydroxythiols (X═OH) make the surface hydrophilic. Titratable terminalgroups (e.g., X═COO or X═NH₃ ⁺) confer hydrophilicity and charge thesurface, which varies as a function of pH. Some other examples are:amino, aldehyde, hydrazide, carbonyl or vinyl groups.

The reaction occurring when these or other groups are used for couplingvarious types of sample molecules are well known from the literature.More complex thiols can be used to serve special functions. For example,polyethylene-glycol-therminated thiols, biotyinylated thiols orthoalkanes bearing metal chelating groups allow the immobilization ofhistidine-tagged proteins and lipids to metal surfaces. More complexsurfaces can easily be created by co-adsorbing two or more thiols withdifferent functional groups.

Turning to an example of chemical surface modification, FIG. 8 depicts aPWI sensor 30 that includes a prism 32 upon which a layer of metal 34 isdisposed. Below the metal layer 34 is a thick dielectric material 36,thus forming a basic PWI device. Additionally, disposed upon dielectric36 is a very thin layer of metal 38 (e.g. gold). The layer of gold 38has been chemically modified with molecules 40 and 42 (which are, inthis case, molecules of tetradecanethiol and 11-mercaptoundecanoic acid,respectively). Molecules 40 may act as sensing molecules (for example,to bind a hydrophobic sample molecule, such as a phospholipid). Molecule42 may provide a basal layer for further functionalization of sensor 30(e.g., by binding a bi- or polyfunctional hydrophilic sensingmolecules).

Employing an oxide as the dielectric layer conveys the advantage ofintrinsic hydrophilicity. Thus, for certain applications (e.g., forself-assembled bilayer formation, see above) it need not be chemicallymodified. If functionalization is desired, silane chemistry, which iswell known in the chemical literature, is the most appropriate approach(see, for example, Bhatia, Suresh K. Analytical Biochemistry (1989) 178,408-413 (1989). Thus, antibodies can be immobilized on the dielectricSiO₂, for example. In principle, surface properties can be controlledusing silane monolayers in the same way as with the thiol-metalchemistry described above.

The invention thus relates to a replaceable sensor unit which is to beused in systems based on many spectroscopy techniques. Each sensingsurface contains at least one functional group, with sensor units foruse in high-throughput screening having different functional groups orligands for interaction with a plurality of molecules present in thesample to be analyzed. The invention moreover relates to processes forfurther functionalization of these sensing surfaces by binding to thembi- or polyfunctional ligands which will interact with molecules in thesample when the measuring operation is taking place.

Various changes in the details and components that have been describedmay be made by those skilled in the art within the principles and scopeof the invention herein described in the specification and defined inthe appended claims. Therefore, while the present invention has beenshown and described herein in what is believed to be the most practicaland preferred embodiments, it is recognized that departures can be madetherefrom within the scope of the invention, which is not to be limitedto the details disclosed herein but is to be accorded the full scope ofthe claims so as to embrace any and all equivalent processes anddevices.

1. A spectroscopy system utilizing a light incident to an interface witha dielectric layer for measuring properties of molecules at surfaces andinterfaces, comprising: a plasmon waveguide sensor including an entrantmedium and said dielectric layer, wherein said dielectric layer is of anoptical thickness selected to produce an observable interference effectfor an incident angle below a critical angle at an interface with thedielectric layer.
 2. The system of claim 1, wherein said dielectriclayer is between 10 and 1000 micrometers in thickness.
 3. The system ofclaim 1, wherein said observable interference effect is measured both intransmittance and reflectance.
 4. The system of claim 1, wherein thesensor further includes a metal or semiconductor layer disposed betweensaid entrant medium and said dielectric layer.
 5. The system of claim 1,wherein the sensor further includes a metal or semiconductor layerdisposed at an interface with said dielectric layer.
 6. The system ofclaim 1, wherein said sensor further includes at least one sensing areaformed at an interface with an emergent medium, said at least onesensing area having undergone functionalization such that a targetchemical within a sample material is immobilized.
 7. The system of claim6, wherein said at least one sensing area comprises a layer of anorganic polymer and at least one functional group supporting bi- orpolyfunctional sensing molecules.
 8. The system of claim 7, wherein saidat least one functional group is selected from the group consisting ofhydroxyl, carboxyl, amino, aldehyde, hydrazide, carbonyl, and vinyl. 9.The system of claim 7, wherein said layer of an organic polymercomprises a polysaccharide.
 10. The system of claim 7, wherein saidfunctional group is selected from one or more members of the groupconsisting of ion exchanger groups, metal chelating groups, andreceptors for biological molecules.
 11. The system of claim 4, whereinsaid sensor further includes at least one sensing area formed at aninterface with an emergent medium, said at least one sensing area havingundergone functionalization such that a target chemical within a samplematerial is immobilized.
 12. The system of claim 11, wherein said atleast one sensing area comprises a layer of an organic polymer and atleast one functional group supporting bi- or polyfunctional sensingmolecules.
 13. The system of claim 12, wherein said at least onefunctional group is selected from the group consisting of hydroxyl,carboxyl, amino, aldehyde, hydrazide, carbonyl, and vinyl.
 14. Thesystem of claim 12, wherein said layer of an organic polymer comprises apolysaccharide.
 15. The system of claim 12, wherein said functionalgroup is selected from one or more members of the group consisting ofion exchanger groups, metal chelating groups, and receptors forbiological molecules.
 16. In a coupled plasmon-waveguide resonancespectroscopic device, wherein a surface plasmon is excited by a lightbeam and propagated along a metallic or semiconductor film to measure aproperty of a sample material placed at an interface of an emergentmedium, the improvement comprising a dielectric layer inserted betweensaid film and said emergent medium, wherein said dielectric layer is ofan optical thickness selected to produce an observable interferenceeffect for an incident angle of light below a critical angle at aninterface with the dielectric layer.
 17. The device of claim 16, whereinsaid dielectric layer is between 10 and 1000 micrometers in thickness.18. The device of claim 16, wherein said observable interference effectis measured both in transmittance and reflectance.
 19. The device ofclaim 16, further including at least one sensing area formed at saidinterface with the emergent medium, said at least one sensing areahaving undergone functionalization such that a target chemical withinsaid sample material is immobilized.
 20. The device of claim 19, whereinsaid at least one sensing area comprises a layer of an organic polymerand at least one functional group supporting bi- or polyfunctionalsensing molecules.
 21. The device of claim 20, wherein said at least onefunctional group is selected from the group consisting of hydroxyl,carboxyl, amino, aldehyde, hydrazide, carbonyl, and vinyl.
 22. Thedevice of claim 20, wherein said layer of an organic polymer comprises apolysaccharide.
 23. The sensor of claim 20, wherein said functionalgroup is selected from one or more members of the group consisting ofion exchanger groups, metal chelating groups, and receptors forbiological molecules.
 24. A method for measuring a property of a samplematerial present at an interface of an emerging medium in aspectroscopic device that includes an interface with a dielectricmember, comprising the following steps: (a) selecting the dielectricmember with an optical thickness capable of producing an observableinterference effect within the dielectric member for an incident angleof light below a critical angle of total internal reflection at saidinterface with the dielectric member; (b) placing said sample materialat said interface of the emerging medium of the spectroscopic device;and (c) performing spectroscopic measurements with light having anincident angle below said critical angle of total internal reflection.25. The method of claim 24, wherein said dielectric member of steps (a)and (b) is between 10 and 1000 micrometers in thickness.
 26. The methodof claim 24, wherein said observable interference effect is measuredboth in transmittance and reflectance.
 27. A method for measuring aproperty of a sample material present at an interface of an emergingmedium in a surface-plasmon spectroscopic device, wherein a surfaceplasmon is excited by a light beam and propagated along a metallic orsemiconductor film, comprising the following steps: (a) coating saidfilm with a dielectric layer being of an optical thickness selected toproduce an observable interference effect within the dielectric memberfor an incident angle of light below a critical angle of total internalreflection at an interface with said dielectric layer; (b) placing saidsample material at said interface of the emerging medium of thesurface-plasmon-resonance spectroscopic device; and (c) performingspectroscopic measurements with light having an incident angle between 0and 90 degrees.
 28. The method of claim 27, wherein said dielectricmember of steps (a) and (b) is between 10 and 1000 micrometers inthickness.